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In this study, a surface modification strategy using natural biopolymers on titanium is proposed to improve bone healing and promote rapid and successful osseointegration of orthopedic implants. Titania nanotubes were fabricated via an anodization process and the surfaces were further modified with polyelectrolyte multilayers (PEMs) based on Tanfloc (a cationic tannin derivative) and glycosaminoglycans (heparin and hyaluronic acid). Scanning electron microscopy (SEM), water contact angle measurements, and X-ray photoelectron spectroscopy were used to characterize the surfaces. Adipose-derived stem cells (ADSCs) were seeded on the surfaces, and the cell viability, adhesion, and proliferation were investigated. Osteogenesis was induced and osteogenic differentiation of human ADSCs on the surfaces was evaluated via mineralization and protein expression assays, immunofluorescent staining, and SEM. The Tanfloc/heparin PEMs on titania nanotubes improved the rate of osteogenic differentiation of ADSCs as well as the bone mineral deposition, and is therefore a promising approach for use in orthopedic implants., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biomaterial-associated thrombus formation and bacterial infection remain major challenges for blood-contacting devices. For decades, titanium-based implants have been largely used for different medical applications. However, titanium can neither suppress blood coagulation, nor prevent bacterial infections. To address these challenges, tanfloc/heparin polyelectrolyte multilayers on titania nanotubes array surfaces (NT) were developed. The surfaces were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and water contact angle measurements. To evaluate the hemocompatibility of the surfaces, fibrinogen adsorption, Factor XII activation, and platelet adhesion and activation were analyzed. The antibacterial activity was investigated against Gram-negative P. aeruginosa and Gram-positive S. aureus. Bacterial adhesion and morphology, as well as biofilm formation, were analyzed using fluorescence microscopy and SEM. The anti-thrombogenic properties of the surfaces were demonstrated by significant decreases in fibrinogen adsorption, Factor XII activation, and platelet adhesion and activation. Modifying NT with tanfloc/heparin also reduces the adhesion and proliferation of P. aeruginosa and S. aureus bacteria after 24 hr of incubation, with no biofilm formation. The modified NT surfaces with tanfloc/heparin polyelectrolyte multilayers are a promising biomaterial for use on implant surfaces because of their enhanced blood biocompatibility and antibacterial properties., (© 2020 Wiley Periodicals, Inc.)

Bone allografts have very limited healing leading to high rates of failure from non-union, fracture, and infection. The limited healing of bone allografts is due in large part to devitalization and removal of the periosteum, which removes osteogenic cells and osteoinductive signals. Here we report techniques for directly coating cortical bone with tissue scaffolds, and evaluate the scaffolds' capacity to support osteoprogenitor cells. Three types of coatings are investigated: N,N,N-trimethyl chitosan-heparin polyelectrolyte multilayers, freeze-dried porous chitosan foam coatings, and electrospun chitosan nanofibers. The freeze-dried and electrospun scaffolds are also further modified with polyelectrolyte multilayers. All of the scaffolds are durable to subsequent aqueous processing, and are cytocompatible with adipose-derived stem cells. Alkaline phosphatase and receptor activator of nuclear factor kappa-B ligand expression at days 7 and 21 suggest that these scaffolds support an osteoprogenitor phenotype. These scaffolds could serve as periosteum mimics, deliver osteoprogenitor cells, and improve bone allograft healing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cortical bone allografts suffer from high rates of failure due to poor integration with host tissue, leading to non-union, fracture, and infection following secondary procedures. Here, we report a method for modifying the surfaces of cortical bone with coatings that have biological functions that may help overcome these challenges. These chitosan-heparin coatings promote mesenchymal stem cell attachment and have significant antibacterial activity against both S. aureus and E. coli. Furthermore, their chemistry is similar to coatings we have reported on previously, which effectively stabilize and deliver heparin-binding growth factors. These coatings have potential as synthetic periosteum for improving bone allograft outcomes., (Copyright © 2012 Wiley Periodicals, Inc.)

Here we present a novel matrix-mimetic nanoassembly based on polysaccharides. Chitosan electrospun fiber networks are decorated with heparin-containing polyelectrolyte complex nanoparticles (PCNs) that present basic fibroblast growth factor (FGF-2), both stably adsorbed to the surfaces and released into solution. These FGF-2/PCN complexes can be released from the fibers with zero-order kinetics over a period of 30 days. Further modification of fibers with a single bilayer of polyelectrolyte multilayer (PEM) composed of N,N,N-trimethyl chitosan and heparin completely prevent release, and the FGF-2/PCN complexes are retained on the fibers for the duration of the release experiment (30 days). We also compare the mitogenic activity of these FGF-2/PCN complexes delivered in two different states: adsorbed to a surface and dissolved in solution. FGF-2/PCN complexes exhibit mitogenic activity with respect to ovine bone marrow-derived mesenchymal stem cells, even after being preconditioned by incubating for 27 days at 37°C in solution. However, when the FGF-2/PCN complexes are adsorbed to chitosan and coated with PEMs, the mitogenic activity of the FGF-2 steadily decreases with increasing preconditioning time. This work demonstrates a new system for stabilizing and controlling the delivery of heparin-binding growth factors, using polysaccharide-based matrix-mimetic nanomaterials. This work also contributes to our understanding of the preferred mode of growth factor delivery from porous scaffolds., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A new method is presented for functionalizing electrospun nanofibers with GAGs and growth factors by PEM deposition. Electrospun chitosan nanofibers, spun from trifluoroacetic acid and dichloromethane, were coated with PEMs, using the polysaccharides heparin and N,N,N-trimethyl chitosan. FGF-2 was adsorbed on the PEM-coated nanofibers. Nanofiber neutralization, PEM construction, and FGF-2 adsorption were monitored using FT-IR spectroscopy and X-ray photoelectron spectroscopy. Alcian blue staining was used to confirm the presence of heparin. SEM was used to study nanofiber morphology., (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It is generally accepted that both surface chemistry and biochemical cues affect mesenchymal stem cell (MSC) proliferation and differentiation. Several growth factors that have strong influences on MSC behavior bind to glycosaminoglycans in interactions that affect their stability and their biochemical activity. The goal of this work was to develop polysaccharide-based polyelectrolyte multilayers (PEMs) to bind and stabilize growth factors for delivery to MSCs. Using the naturally derived polysaccharides chitosan and heparin, PEMs were constructed on gold-coated glass chips, tissue-culture polystyrene (TCPS), and titanium. PEM construction and basic fibroblast growth factor (FGF-2) adsorption to PEMs were evaluated by Fourier transform surface plasmon resonance, X-ray photoelectron spectroscopy, and polarization modulation infrared reflection absorption spectroscopy. The functional response of bone marrow-derived ovine MSCs to FGF-2 on PEM-coated TCPS and titanium was evaluated in vitro, in the presence and absence of adsorbed fibronectin. The effect of FGF-2 dose and presentation on MSC attachment and proliferation was evaluated using low-serum media, over four days. On PEM-coated TCPS, we found that FGF-2 adsorbed to heparin-terminated PEMs with adsorbed fibronectin induces greater cell density and a higher proliferation rate of MSCs than any of the other conditions tested, including delivery of the FGF-2 in solution, at an optimally mitogenic dose. Cell densities on day four were 1.8 times higher when FGF-2 was delivered by adsorption to the PEM than when FGF-2 was delivered in solution. This system represents a promising candidate for the development of surface coatings that can stabilize and potentiate the activity of growth factors for therapeutic applications. Interestingly, the same effects were not observed when FGF-2 was delivered by adsorption to PEMs on titanium. When the polysaccharide-based PEMs were formed on titanium, the proliferative response of ovine MSCs to adsorbed FGF-2 was not as strong as the response to FGF-2 delivered in solution.

The formation of polyelectrolyte complex nanoparticles (PCN) was investigated at different charge mixing ratios for the chitosan-heparin (chi-hep) and chitosan-hyaluronan (chi-ha) polycation-polyanion pairs. The range of 0.08-19.2 for charge mixing ratio (n(+)/n(-)) was examined. The one-shot addition of polycation and polyanion solutions used for the formation of the PCN permitted formation of both cationic and anionic particles from both polysaccharide pairs. The influence of the charge mixing ratio on the size and zeta potential of the particles was investigated. The morphology and stability of the particles when adsorbed to surfaces was studied by scanning electron microscopy (SEM). For most conditions studied, colloidally stable, nonstoichiometric PCN were formed in solution. However, PCN formation was inhibited by flocculation at charge mixing ratios near 1. When adsorbed to surfaces and dried, some formulations resulted in discrete nanoparticles, while others partially or completely aggregated or coalesced, leading to different surface morphologies.

The goal of this work is to explore the effects of solution ionic strength and pH on polyelectrolyte multilayer (PEM) assembly, using biologically derived polysaccharides as the polyelectrolytes. We used the layer-by-layer (LBL) technique to assemble PEM of the polysaccharides heparin (a strong polyanion) and chitosan (a weak polycation) and characterized the sensitivity of the PEM composition and layer thickness to changes in processing parameters. Fourier-transform surface plasmon resonance (FT-SPR) and spectroscopic ellipsometry provided in situ and ex situ measurements of the PEM thickness, respectively. Vibrational spectroscopy and X-ray photoelectron spectroscopy (XPS) provided details of the chemistry (i.e., composition, electrostatic interactions) of the PEM. We found that when PEM were assembled from 0.2 M buffer, the PEM thickness could be increased from less than 2 nm per bilayer to greater than 4 nm per bilayer by changing the solution pH; higher and lower ionic strength buffer solutions resulted in narrower ranges of accessible thickness. Molar composition of the PEM was not very sensitive to solution pH or ionic strength, but pH did affect the interactions between the sulfonates in heparin and amines in chitosan when PEM were assembled from 0.2 M buffer. Changes in the PEM thickness with pH and ionic strength can be interpreted through descriptions of the charge density and conformation of the polyelectrolyte chains in solution.

Incomplete osseointegration is primary cause of failure for orthopedic implants. New biomaterials that present stable signals promoting osteogenesis could reduce failure rates of orthopedic implants. In this study bone morphogenetic protein‐2 (BMP‐2) was delivered from titania nanotubes (Nt) modified with chitosan/heparin polyelectrolyte multilayers (PEMs). The surfaces were characterized by scanning electron microscopy and X‐ray photoelectron spectroscopy. BMP‐2 release from the surfaces was measured in vitro for up to 28 days. After an initial burst release of BMP‐2 during the first 2 days, most of the BMP‐2 remained on the surface. To determine the osteogenic properties of these surfaces, they were seeded with rat bone marrow cells; alkaline phosphatase (ALP) activity, total protein, calcium deposition, and osteocalcin were measured up to 4 weeks in vitro. When compared to Nt surfaces, the surfaces with BMP‐2 induce greater osteocalcin and calcium deposition. PEMs provide sustained presentation of BMP‐2, from a biomimetic surface. This enhances the osteogenic properties of the surface without requiring supraphysiologic growth factor dose. This growth factor delivery strategy could be used to improve bone healing outcomes and reduce complications for recipients of orthopedic implants. [ABSTRACT FROM AUTHOR]

The direct delivery of growth factorsto sites of tissue healingis complicated by their relative instability. In many tissues, theglycosaminoglycan (GAG) side chains of proteoglycans like aggrecanstabilize growth factors in the pericellular and extracellular space,creating a local reservoir that can be accessed during a wound healingresponse. GAGs also regulate growth factor-receptor interactions atthe cell surface. Here we report the development of nanoparticlesfor growth factor delivery that mimic the size, GAG composition, andgrowth factor binding and stabilization of aggrecan. The aggrecan-mimeticnanoparticles are easy to assemble, and their structure and compositioncan be readily tuned to alter their physical and biological properties.We use basic fibroblast growth factor (FGF-2) as a model heparin-bindinggrowth factor, demonstrating that aggrecan-mimetic nanoparticles canpreserve its activity for more than three weeks. We evaluate FGF-2activity by measuring both the proliferation and metabolic activityof bone marrow stromal cells to demonstrate that chondroitin sulfate-basedaggrecan mimics are as effective as aggrecan, and heparin-based aggrecanmimics are superior to aggrecan as delivery vehicles for FGF-2. [ABSTRACT FROM AUTHOR]

Proteoglycans have vital biochemical and biomechanical functions. Their proteolytic degradation results in loss of these functions. We have previously reported nonprotein proteoglycan-mimetic graft copolymers that stabilize and deliver growth factors and are not subject to proteases. Here we expand our investigation of these proteoglycan mimics by also investigating their effects on hydrogel mechanical properties. Four polysaccharide side chains, chondroitin sulfate, heparin, dextran, and dextran sulfate, are each grafted to a hyaluronan backbone. The polysaccharides and graft copolymers are added to agarose hydrogels. Cyclic compression and stress relaxation tests reveal how the addition of the polysaccharides and graft copolymers influence hydrogel modulus. Cells encapsulated in agarose hydrogels containing chondroitin sulfate and the chondroitin sulfate graft copolymer have decreased cell viability and metabolic activity compared to cells in unmodified agarose hydrogels. These multifunctional additives can be used to improve both the biochemistry and biomechanics of materials, warranting further optimization to overcome the potentially negative effects these may have on cell viability and activity. [ABSTRACT FROM AUTHOR]

We present chitosan (CHT)/heparin (HP) polyelectrolyte complexes (PECs) that quickly adsorb citrate-capped silver nanoparticles (AgNPs). CHT/HP blends in ionic liquid ([HMIm][HSO 4 ]) form durable PECs after precipitation in water. CHT/HP PECs have positive Zeta potentials (higher than +20 mV). They adsorb citrate-capped AgNPs (Zeta potential of - 12.25 mV) synthesized from Turkevich's method. PEC/AgNPs composites are characterized by spectroscopic, thermal, and microscopy analyses. AgNPs on the PEC surfaces are confirmed by transmission electron microscopy. PECs adsorb AgNPs from aqueous suspensions, achieving ≈ 95% of removal (17.18 μg of AgNPs per milligram of PEC) after only 10 min. The pseudo-second-order kinetic model adjusted well to the experimental data. The PECs release approximately 11.80 μg/mg Ag+ (66%) compared to the initial adsorbed AgNPs content (17.18 μg/mg) after 7200 min at pH 2.0. The PECs present low swelling degrees (between 130 and 150%), supporting high stability in water. PEC/AgNPs composites promote significant bactericidal activity toward Staphylococcus aureus and Escherichia coli between 0.25 and 0.5 mg/mL. This study shows a new strategy to create hybrid polysaccharide/AgNPs composites. PECs can stabilize the AgNPs and release Ag+ ions, supporting antimicrobial materials. • Polyelectrolyte complexes (PECs) were synthesized using a green solvent. • 95% of the citrate-capped silver nanoparticles were adsorbed on PECs' surface. • Citrate-capped silver nanoparticles are visible on PECs. • Hybrid materials present low heparin release at simulated fluids. • The Citrate-capped silver nanoparticles sorption is faster than the Ag+ ion release. [ABSTRACT FROM AUTHOR]